C–C2Similarly, THC exposure misrouted hippocampal CB1R+ afferents, significantly increasing their density in the CA1 stratum radiatum [blue in (C) and (C1)(* indicates the position of neuronal somata)]. Quantitative densitometry of CB1R+ boutons in the stratum radiatum is shown in (C2).

F. Rewiring and reduced synaptic plasticity in the cortical circuitry were not associated with long‐lasting modifications of synaptic protein expression in the hippocampus of offspring prenatally exposed to THC (P120). Fold changes were normalized to gapdh and expressed as percentages (n =4/group; see Supplementary Fig S1B and C).

D. WIN55,212‐2 induced Erk1/2 phosphorylation to a similar degree in vehicle‐ and THC‐treated offspring, suggesting that in vivo THC administration in the present experimental paradigm did not desensitize CB1Rs. Representative Western blots from duplicate experiments are shown.

Data information: Data were expressed as means ± s.e.m.; ***P <0.001, *P <0.05. Scale bars, 100 μm (A,B). For a lits of abbreviations, see the Supplementary information.Source data are available online for this figure.

SCG10 is expected to be broadly expressed during corticogenesis, coincident with the onset of THC administration, if its loss is to underpin THC‐induced modifications of axonal extension. Indeed, we detected SCG10 mRNA by E14 with its expression level gradually increasing until birth (Fig 3B2). SCG10 mapped to long‐range forebrain projections (Supplementary Fig S3B–D), was enriched in growth cone‐like structures (Fig 3C), and co‐distributed with CB1Rs in both the intermediate zone of the cerebral cortex (Fig 3D–D2) and the primordial hippocampus (Supplementary Fig S2C and C1). CB1Rs are expressed during the radial migration and morphogenesis of pyramidal cells in the cerebral cortex (Mulder et al, 2008). Here, we validated SCG10 localization by showing its enrichment in cells with morphologies reminiscent of pyramidal cells in the cortical plate (Westerlund et al, 2011) (Fig 3D and D1). SCG10 is localized in the cytosol (Grenningloh et al, 2004). By using high‐resolution laser scanning microscopy we show that approximately 40% of SCG10+ and CB1R+ puncta are closely associated, with approximately 3% directly overlapping in corticofugal axons (Fig 3D2 and D3). This finding establishes that SCG10 is proximal to CB1Rs in corticofugal axons, and could be a downstream target of this GPCR in particular neurite domains. These data, together with retained cerebral SCG10 mRNA and protein expression postnatally in the brain of THC‐exposed fetuses (Supplementary Fig S1G and G1), highlight SCG10 as a developmentally regulated candidate protein whose loss can confer THC effects on neuronal morphology.

A. Overview of organotypic slices prepared from E14.5 mouse embryos. Note the retained anatomical organization, as well as CB1R and SCG10 expression patterns of the cerebral cortex and thalamus. L1‐NCAM was used as an axonal marker throughout.

If the CB1R‐JNK‐SCG10 pathway alone is sufficient to account for the THC‐induced cytoarchitectural modifications of cortical neurons then overexpression of a functionally inactive, pseudophosphorylated SCG10 mutant (Tararuk et al, 2006; Westerlund et al, 2011) (i.e. aspartate substitution of Ser62 and Ser73 (SCG10‐DD)) would phenocopy THC effects. Consistent with this hypothesis, SCG10‐DD overexpression appeared to outcompete endogenous SCG10 in neurites (Fig 7A), reduced neurite outgrowth and occluded THC‐induced modifications to axonal morphology (84.0 ± 8.7 μm [THC+EGFP‐C1] versus 92.6 ± 9.6 μm [THC+SCG10‐DD], P >0.5; Fig 7A and A1). PC12 cells transfected with SCG10‐DD for 24‐72 h were used to show that SCG10‐DD is overexpressed at the expense of endogenous (wild‐type) SCG10. By Western blotting PC12 cell lysates, we separated GFP‐tagged SCG10‐DD from unlabeled endogenous SCG10 and found the progressive reduction of the latter SCG10 form as a factor of time (Fig 7A2). In addition, siRNA‐mediated knockdown of SCG10 in cultured cortical neurons reduced neurite outgrowth (Fig 7B–B2) to an extent similar to that seen upon SCG10‐DD overexpression. Overall, these data highlight the central role of SCG10 degradation in axonal growth defects imposed by prenatal THC exposure.

CB1Rs are predominantly presynaptic in the adult brain (Kano et al, 2009). Likewise, cell‐surface CB1Rs are only found on the axons of developing neurons (McDonald et al, 2007). The domain‐specific cell‐surface targeting of CB1Rs allowed us to assay whether the molecular pathway we uncovered is specific to elongating axons. To this end, we first determined THC‐induced changes to SCG10 and acetylated tubulin contents in secondary, short neurites lacking CB1Rs, putative primordial dendrites (Supplementary Fig S6C). THC induced tubulin acetylation in both the growth cones and neurite stems (P <0.05 versus vehicle‐treated control; Supplementary Fig S6C1 and C2). In contrast, SCG10 levels remained unchanged (Supplementary Fig S6C3 and C4). Next, we co‐localized acetylated tubulin and post‐synaptic density protein 95 (PSD95), a marker of excitatory post‐synapses (Tomasoni et al, 2013), to dissect THC effects at established synapses. THC did not induce tubulin acetylation in either PSD95+ post‐synapses or apposing pre‐synaptic terminals (Supplementary Fig S6D–D4). In sum, these experiments establish the axonal specificity of CB1R‐dependent SCG10 degradation, and specifically implicate this mechanism in the modulation of axonal growth and guidance.

Discussion

Our results, for the first time, define a specific molecular target for THC in the developing central nervous system, whose modifications can directly and permanently impair the wiring diagram of neuronal networks during corticogenesis. These finding have direct and increasing human relevance since selective cultivation of Cannabis subspecies recently significantly altered their phytocannabinoid contents, with particularly pronounced increases in THC content (Pitts et al, 1992; Pijlman et al, 2005). We first mount compelling experimental support to the hypothesis that, when available for prolonged periods in vivo, THC disrupts endocannabinoid signaling at their cognate CB1R. Our mRNA and protein profiling of molecular components controlling 2‐AG metabolism suggest that THC not only can act as a “functional antagonist” (i.e. displacement of endocannabinoid binding to the CB1R), but can disrupt 2‐AG signaling by reducing both CB1R and DAGLα expression during cortical development. We attribute the discrepancy between MGL mRNA and protein expression levels to MGL being prone to posttranslational modifications facilitating its proteasomal degradation (Keimpema et al, 2013). Next, we identify SCG10 as a novel signaling node of morphogenic CB1R signaling since (i) SCG10 is ideally poised to link cell‐surface CB1R activation and pleiotropic downstream mitogen‐activated protein kinase (particularly JNK) activation (Rueda et al, 2000; Berghuis et al, 2007; Keimpema et al, 2011) to cytoskeletal instability (Keimpema et al, 2011), and (ii) the rapid degradation of SCG10 can allow ectopic growth with ensuing modifications to axodendritic morphology in vitro recapitulating corticofugal growth defects in vivo. This mechanism appears to be restricted to the THC‐induced reorganization of axonal morphology, which is compatible with the sole localization of CB1Rs to growth cones and presynapses in the developing (Keimpema et al, 2010) and adult nervous systems (Kano et al, 2009), respectively. Nevertheless, we recognize that CB1R activation might engage multiple co‐existent signaling pathways (e.g. RhoGTPases (Berghuis et al, 2007; Nithipatikom et al, 2012)) to couple cytoskeletal reorganization to the orchestration of axonal morphology.

THC exposure altered the levels of 35 proteins in the fetal cerebrum. Previously, a total of 49 differentially‐regulated genes were identified by cDNA microarrays in nervous tissues from adult rats treated with THC for 24 h, 7 or 21 days (Kittler et al, 2000), including molecular constituents of endocannabinoid and lipid biosynthesis, and signal transduction machineries. Conspicuously, neural cell adhesion molecule and myelin basic protein, implicated in axonal growth (Yuan et al, 2003; Keimpema et al, 2011) and myelination by oligodendrocytes were also altered. Here, our protein profiling by mass spectrometry combined with cell biology provides a new perspective on developmentally‐regulated candidates, and suggests that the reorganization of synaptic structure and plasticity is an inherent feature of THC action (Keimpema et al, 2011). This is further supported by the observation that SCG10 expression prevails during adulthood and aging in neuronal clusters with long‐lasting synaptic plasticity (Peng et al, 2004). In using iTRAQ for quantitative candidate discovery we reasoned that mRNA may not be translated into biologically active protein. Moreover, we conceptually approached the 17% decrease in cortical SCG10 amount as an indication that a subset of cortical neurons selectively, rather than all of them indiscriminately, down‐regulated SCG10 protein expression. Indeed, SCG10 is primarily expressed in a contingent of neurons in the fetal cortical plate that adopted pyramidal cell‐like positioning and axodendritic morphology. Linking SCG10 deregulation to pyramidal cell development is not unexpected since genetic deletion of CB1Rs in pyramidal cells destined to superficial cortical layers leads to axon fasciculation and targeting errors (Mulder et al, 2008). Moreover, since CB1Rs are not expressed in cortical proliferative zones (Goncalves et al, 2008; Mulder et al, 2008), CB1R‐mediated adverse THC effects will likely impact cortical wiring rather than neuronal diversification in the fetal cerebrum. Nevertheless, we find similarities in the molecular identity and functions of THC's differentially regulated targets in developmental and adult settings (Kittler et al, 2000), such as proteins implicated in secondary metabolism, protein folding and signal transduction (Supplementary Fig S3A). Our data in conjunction with large‐scale analysis from adult brain (Kittler et al, 2000) reinforce that THC's mode of action involves the disrupted development and/or postnatal maintenance of synapses critical for highly ordered executive and cognitive functions.

Recreational cannabis use is particularly prevalent in the young adult age group, including women of child‐bearing age (Substance Abuse & Mental Health Service Administration, 2010). Although recent population statistics revealed significant fluctuations due to geoeconomic variables, > 10% of pregnancies in US and Europe are associated with maternal cannabis exposure (Substance Abuse & Mental Health Service Administration, 2010). Cannabis spp. reportedly contain > 400 bioactive components, with THC being its primary psychoactive constituent. During the past decades, selective agriculture of Cannabis spp. resulted in increased THC content at the expense of cannabidiol (Pitts et al, 1992; Pijlman et al, 2005). In the context of the present study, this is of particular concern since we predict that higher THC concentrations will be, upon efficient cross‐placental transfer (Grotenhermen, 2003), increasingly detrimental for fetal development and postnatal health. Therefore, irrespective of the legal status of cannabis, caution must be exercised to hinder fetal cannabis exposure due to its unequivical impact on the establishment of synaptic connectivity in neuronal networks underpinning memory encoding, cognition and executive skills. Moreover, abnormal synaptic organization, even if remaining latent for long periods, might be prone to “circuit failure” if provoked. A “double hit” scenario of cortical failure when a labile network advances into a runaway cascade upon a secondary insult therefore might account for the increased incidence of schizophrenia, depression and addiction in offspring prenatally exposed to cannabis (Substance Abuse & Mental Health Service Administration, 2010; Keimpema et al, 2011).

Materials and Methods

Animals

Tissues from fetal (E14.5–E18.5), neonatal (P10) and adult (P120) wild‐type and CB1R−/− mice were processed as described (Berghuis et al, 2007; Keimpema et al, 2010). Wild‐type and CB1R−/− pregnant mice on C57Bl/6 background (Monory et al, 2006) were injected intraperitoneally with THC (3 mg/kg, THC Pharm) (Mato et al, 2004; Campolongo et al, 2007), WIN55,212‐2 (5 mg/kg, Tocris) or AM 251 (5 mg/kg, Tocris) from E5.5 to E17.5 daily. Embryos were harvested on E18.5, their brains immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PFA‐PB), cryosectioned (16 μm) and glass‐mounted coronal sections prepared for histochemistry (Keimpema et al, 2010). Postnatal offspring were perfusion‐fixed (PFA‐PB), their brains cryosectioned (40 μm) in the coronal plane, and processed as free‐floating sections (Mulder et al, 2011). Given their preferential sensitivity to cannabis (Spano et al, 2007), male offspring were analyzed. JNK1−/− mice were produced as described (Dong et al, 1998) and bred conventionally (University Hospital of Schleswig‐Holstein). We used JNK1−/− mice back‐crossed for 10 generations onto a C57Bl/6N background, which served as controls. Where relevant, mouse genotyping was performed by amplifying genomic DNA using primer sets allowing the discrimination of the endogenous locus from targeted alleles. Experimental protocols on live animal conformed to the European Communities Council Directive (86/609/EEC) and were approved by the Home Office of the United Kingdom, as well as the Ministerium für Landwirtschaft und Naturschutz, Kiel, Germany. Efforts were made to minimize the number of animals and their suffering throughout the experiments.

Cortical cultures, transient transfection and siRNA knockdown

Cultured neurons were isolated from E16.5 mouse cortices (Keimpema et al, 2010). Neurons for morphological analysis were plated (25,000/well) onto poly‐D‐lysine‐coated coverslips. On day 2 in vitro (DIV), neurons were stimulated by either 500 nM or 2 μM THC for 24 h. THC at 10 μM concentration was used in acute experiments (10 min). Cells were immersion fixed in PFA‐PB for 20 min.

For transient transfection, neurons were seeded at a density of 50,000 cells/well, and transfected with EGFP‐tagged SCG10S62D/S73D (SCG10‐DD) or pEGFP‐C1 (control) using Lipofectamine 2000 transfection agent (Invitrogen, for 6 h) 24 h later. THC (2 μM) stimulation for 24 h was carried out 1 day later. For the biochemical analysis of wild‐type versus SCG10‐DD contents, PC12 cells (500,000/well) were transfected with the constructs as above, lysed at the time points indicated in Fig 7A2, and probed for SCG10 and GFP. For siRNA knockdown of SCG10, cortical neurons (50,000 cells/well, 24‐well format) were grown in DMEM/F12 (1:1) containing B‐27 supplement (2%) and L‐glutamine (2 mM) for 24 h. Subsequently, cultures were co‐transfected with a GFP construct (0.5 μg; pEGFP‐C1) used to identify positive transfectants, and either scrambled siRNAs (50 pmol; D‐001810‐10‐05; Thermo Scientific) or a pool of SCG10 siRNAs (50 pmol; L‐045412‐01‐0005; Thermo Scientific) with Lipofectamine 2000 (Invitrogen) for 45 min. Cells were washed once in full growth medium and returned to their original medium in presence of THC (2 μM) or vehicle for 24 h. Cultures were immersion fixed in 4% PFA‐PB and processed as described above.

Organotypic slice culture

Organotypic cultures were prepared from C57Bl/6 mice on E14.5. Brains were rapidly dissected and subsequently embedded in 5% low‐melt agarose (Promega). Coronal sections (300 μm) were cut on a vibratome (VT1200S, Leica), and transferred onto polytetrafluoroethylene culture membranes (30 mm in diameter; 0.4 μm pore size; Millipore) in 6‐well plates containing DMEM supplemented with glutamine (2 mM), fetal bovine serum (10%) and penicillin‐streptomycin (1%) in a total volume of 1 ml. After 1 h incubation, the medium was replaced with Neurobasal medium also containing glutamine (2 mM), B27 supplement (2%) and penicillin‐streptomycin (1%) and slices allowed to equilibrate overnight. The next day, slices were pre‐treated with SP600125 (5 μM) or vehicle for 2 h. Subsequently, THC (10 μM) was applied alone or in combination with SP600125 (5 μM) for an additional 30 min. Afterwards, slices were fixed with 4% PFA‐PB and processed routinely for multiple‐labeling immunofluorescence histochemistry. High‐resolution images of corticofugal axons in E14.5 slices were captured at 20× primary magnification (Fig 6B). Mean intensities of SCG10 and L1‐NCAM immunoreactive structures were measured with the ZEN2009 software (Zeiss; Fig 6B1). The area coverage of immunoreactive pixels was calculated by expressing the ratio of the immunofluorescent signal and the total surface area (Fig 6B3 and B4). Gray scale values were accepted between 10 and 245 to reduce unspecific noise.

Quantitative morphometry

Multiple immunofluorescence labeling of fetal and postnatal mouse brains and cultured neurons was performed by applying select cocktails of affinity‐purified antibodies (Keimpema et al, 2010; Mulder et al, 2011) (Supplementary Table S1). Particular care was taken to assess the reliability of anti‐SCG10 antisera, including simultaneous detection using two antibodies (from different hosts) raised against non‐overlapping protein epitopes (Supplementary Fig S3D). Quantitative analysis at P10 was aided by acquiring tiled 12‐bit greyscale images at 10× primary magnification on a VSlide slide‐scanning microscope (MetaSystems) equipped with a CoolCube 1 camera. Hoechst 33342 nuclear stain was used to delineate cortical laminae. Alternatively, images were acquired on a Zeiss 710LSM confocal laser‐scanning microscope using spectral detection tuned for maximal signal separation and digital zoom ranging from 1.5 to 3.0× (Keimpema et al, 2010; Mulder et al, 2011). The co‐existence of immunosignals was verified by capturing serial orthogonal z image stacks at 40× primary magnification (up to 3× optical zoom) and accepted if these were present without physical signal separation in ≤1.0‐μm optical slices, and overlapped in all three (x, y and z) dimensions within individual cellular domains. Co‐localization between CB1Rs and SCG10 in vivo was performed at 63× primary magnification and 5× optical zoom (Zeiss 700LSM). The physical distance between individual CB1R+ and SCG10+ puncta was divided in three groups [overlap, contact (i.e. adjacent signals without overlap) and separate; Fig 3D2 and D3], and expressed as the percentage of the total number of immunoreactive puncta counted per axon.

Morphometric analysis of cultured neurons was aided by the ZEN2009 software and included: (i) filopodia number and density (expressed per 100 μm neurite segment), (ii) neurite branching (n), (iii) length of the primary neurite (μm), (iv) the diameter of neurite bundles (μm), (v) the morphology, surface area and filopodia number of growth cones, and (vi) fluorescence intensity distribution of SCG10 in growth cones and along neurite shafts (Keimpema et al, 2010). The longest process emanating from neuronal somata was analyzed and considered as the prospective axon. Similarly, the transverse diameter of L1‐NCAM+ corticofugal axons in fetal wild‐type and CB1R−/− brains after THC or vehicle administration was measured with the ZEN2009 software package on calibrated images. Only first‐order fascicles of > 3 μm in diameter were analyzed. In P10 or P120 animals, synapse density and distribution were measured on high‐resolution graphic images exported into the UTHSCSA ImageTool (version 3.0). CB1R+ “perisomatic baskets”, defined as CB1R+ terminal‐like boutons engulfing MAP2+ somata in layers (L)II/III of the neocortex on at least three quadrants (Fig 1B), were counted in ImageJ 1.45s with their density expressed per 104 μm2. The density of terminal profiles (> 5 continuous pixels at 1670 × 945 pixel input resolution) was expressed per 103 μm2.

The intensity of acetylated tubulin in pre‐ and postsynaptic terminals of cortical cultures (4DIV) was measured by ImageJ 1.45s and expressed as arbitrary units (Supplementary Fig S6D–D3). Postsynaptic terminals were visualized by postsynaptic density protein 95 (PSD95) immunoreactivity and measured if a PSD95− process of another individual neuron (putative pre‐synapse) made contact. The brightness or contrast of confocal laser‐scanning micrographs was occasionally linearly enhanced. Multi‐panel images were assembled in CorelDraw X5.

High‐throughput neurite tracking

Primary cortical neurons were isolated and plated as above. After 10 h, cultures were treated with THC (2 μM) or vehicle for an additional 62 h. Drug treatments were performed in quadruplicates, and imaged live in parallel using an IncuCyte Zoom live‐cell imaging devise (Essen Bioscience). Time‐lapse images were acquired every 2 h. The growth rate of neurites in each well was obtained by measuring the surface area covered by neurites, and expressed as mm/mm2.

Electrophysiology

Transverse coronal slices (300 μm; Leica VT1200S vibratome) of the dorsal hippocampus were prepared in ice‐cold solution (90 NaCl, 2.5 KCl, 1.25 Na2HPO4, 0.5 CaCl2, 8 MgSO4, 26 NaHCO3, 20 D‐Glucose, 10 HEPES, 3 Na‐pyruvate, 5 Na‐ascorbate; in mM) on P120 after rapidly dissecting the brains of decapitated animals (Mulder et al, 2011). Experiments on equilibrated slices (> 60 min; pH7.4 under continuous oxygenation), which were continuously superfused (~10 ml/min) with artificial cerebrospinal fluid (ACSF) and oxygenated at 33–34°C, were performed in a round 1.5‐ml recording chamber. Field excitatory postsynaptic potentials (fEPSPs) from the CA1 subfield were recorded upon orthodromic stimulation of Schaffer collaterals using a voltage stimulus isolator (A360LA, World Precision Instruments) connected to a concentric bipolar platinum‐iridium stimulating electrode (MX211ES, FHC & Co), positioned in the stratum radiatum at the CA2/CA1 border. The impulse intensity was set to 65% of the current that evoked minimum saturated fEPSPs, and delivered at 0.66 Hz. Recording borosilicate glass (Hilgenberg) capillary electrodes (3–4 MΩ, P‐1000, Sutter) filled with ACSF were positioned 500‐700 μm away from the stimulating electrode. One was placed in the str. radiatum of CA1 to sample fEPSPs, while another was inserted in the CA1 str. pyramidale to record population spikes (Supplementary Fig S1E). Both recording electrodes were connected to high input‐impedance headstages (HEKA EPC‐10). After a baseline period of > 10 min, long‐term depression of Schaffer collateral‐CA1 synaptic responses and extracellular potentials was induced by 900 pulses at 1 Hz (at 100% intensity of the test stimulus) with fEPSPs and population spikes recorded simultaneously for ≥ 40 min. Stimulation artifacts were continuously monitored, and recordings with > 20% change in the fiber volley amplitude after LTD induction were excluded. We recorded the paired‐pulse ratio (PPR) with interstimulus intervals of 10, 20, 40, 60, 80, 100, 150 and 200 ms (at 100% intensity of the test stimulus) with recordings made simultaneously in str. radiatum (Fig 1E) and str. pyramidale (Supplementary Fig S1F). The slope of fEPSPs, as well as the amplitude of population spikes was analyzed. N =5–6 mice/group, n =2–3 slices per brain were analyzed. Figure 1D2 was generated by binning data on fEPSP suppression during a 15‐min period of recordings (indicated by orange background, Fig 1D).

iTRAQ proteomics, nLC‐MALDI/MS/MS and nLC‐ESI/MS/MS target discovery

Proteins were extracted from fetal cortices (n =5/3 THC/vehicle from independent pregnancies) by homogenization in triethylammonium bicarbonate (25 mM), Na2CO3 (20 mM) and protease inhibitor cocktail (all from Sigma, pH10), with their concentration determined using the bichinconinic acid (BCA) protein assay (Novagen). Proteins were acetone precipitated, and 100 μg used for isobaric tag for relative and absolute quantitation in 8‐plex layout (iTRAQ, ABSciex). Proteins were denatured, reduced, alkylated, trypsin digested, individually labeled with appropriate iTRAQ tags, pooled, concentrated (SpeedVac, Thermo Scientific), re‐suspended in 1.4 ml load buffer (10 mM KH2PO4 pH 3.0 in 25% acetonitrile) and sonicated. The peptides were then separated by cation exchange chromatography on a PolySulfoethyl A column (PolyLC) over 30 min with a KCl gradient increasing up to 0.5 M, and 0.5 ml fractions collected. Twenty fractions across the elution profile of similar peptide concentration were generated and concentrated (SpeedVac). Fractions were re‐suspended in 0.1% trifluoroacetic acid (TFA) and desalted on C18 spin columns (PepClean C18, Thermo Scientific). For each fraction half of the sample was separated using a Dionex UltiMate 3000 nanoLC (Dionex) equipped with a PepMap100 C18 300 μm × 5 mm trap and 75 μm × 15 cm column (Dionex), using a 3.5 h gradient of increasing acetonitrile concentration, containing 0.05% TFA (5–35% acetonitrile in 3 h, 35–50% in a further 30 min, followed by 95% acetonitrile). The eluent was spotted onto a MALDI target plate, along with α‐cyano‐4‐hydroxycinnamic acid (2 mg/ml in 70% acetonitrile: 0.1% TFA matrix solution) using a Dionex Probot spotter. The nLC‐MALDI/MS/MS runs were analyzed using an 4800 MALDI TOF/TOF Analyser (ABSciex) equipped with a Nd:YAG 355 nm laser in a plate wide data‐dependent manner. All spots were initially analyzed in positive MS mode in the range of 800–4000 m/z by averaging 1,000 laser spots (Shirran & Botting, 2010). The MS ions that satisfied the precursor criteria (200 ppm fraction to fraction precursor exclusion, S/N ratio > 20) were selected for subsequent MS/MS from the spot in where the MS ion gave the highest counts, with up to 5 MS/MS being acquired from each spot, selecting the strongest precursor ion first. MS/MS spectra were acquired with a maximum of 3,000 laser shots or until the accumulated spectrum reached a S/N ratio of 35 for 10 peaks. All MS/MS data were acquired using 1 keV collision energy.

The remaining half of the desalted fractions were separated using an UltiMate nanoLC (Dionex) equipped with a PepMap C18 trap and column, using a gradient of increasing acetonitrile concentration, containing 0.1% formic acid (5–35% acetonitrile in 180 min, 35–50% in a further 30 min, followed by 95% acetonitrile). The eluent was sprayed into a Q‐Star XL tandem mass spectrometer (ABSciex) and analyzed in Information Dependent Acquisition (IDA) mode, performing 1 s of MS followed by 3 s MS/MS analyses of the 2 most intense peaks seen by MS. These masses were then excluded from analysis for the next 60 s. Rolling collision energy was employed for fragmentation, set 10V higher than that normally used for peptides, to provide sufficient peptide fragmentation and generation of the iTRAQ reporter groups.

Mass spectrometric data analysis

For the nLC‐ESI/MS/MS, MS/MS data for doubly and triply charged precursor ions was converted to centroid data, without smoothing, using the Analyst QS1.1 mascot.dll data import filter with default settings. The data files were processed by Mascot v2.2 (Matrix Science). All searches were performed against the NCBI database using a mouse taxonomy filter (137 038 sequences). Automatic isotope correction was carried out using the values supplied with the ABSciex reagents. The “MS/MS averaging of IDA dependents” had a precursor mass tolerance for grouping of 0.1 and the maximum number of cycles between groups and minimum number of cycles per group were both set to 1. The MS/MS settings included: spectra de‐isotoped (except for the iTRAQ reporter region), peak areas reported, spectra rejected if they contained < 10 peaks, and peaks not removed if they were close to the precursor m/z. The nLC‐ESI/MS/MS data were searched with a tolerance of 0.08 Da for the precursor ions and 0.2 Da for the fragment ions. The nLC‐MALDI/MS/MS data were extracted using TS2Mascot 1.0.0 (Matrix Science) and the data saved to a peak list. The nLC‐MALDI/MS/MS data were searched with tolerances of 100 ppm for the precursor ion and 0.5 Da for the fragment ions. For both ionization routes the following settings were used: trypsin was the cleavage enzyme, one missed cleavage, methylthio modification of cysteines and iTRAQ 8‐plex modification of lysines and N‐terminal amines were fixed modifications; methionine oxidation was selected as a variable modification. The following settings were used to manipulate the quantification results: the protein ratio type was the “weighted” geometric mean, there was no normalization, outlier removal was “automatic” (Dixon's method up to 25 data points, Rosner's method above 25 data points), the peptide threshold was “at least homology” (peptide score did not exceed the absolute threshold but was an outlier from the quasi‐normal distribution of random scores), the minimum number of unique peptides was two, and peptides were required to be the top ranking peptide matches. An automatic decoy database search was also performed.

Human subjects, in situ hybridization and Western blotting

Midgestational fetal brain subjects (18–22 weeks of gestation) were collected after saline‐induced elective abortions under Institutional Review Board approval at SUNY Downstate Medical Center, Brooklyn, New York (Hurd et al, 2005). Fetal brains were fixed in 1% PFA‐PB for 24 h, frozen in dry ice‐cooled isopentane, cryosectioned in the coronal plane (20 μm) and stored at −30°C. In situ hybridization to study SCG10 mRNA expression was performed as described (Hurd, 2003). Briefly, PCR‐derived RNA probes spanning exons 2–4 of human SCG10 (NM_001199214; T7‐SCG10 sense primer: CTGTAATACGACTCACTATAGGG‐AGGAGCTGTCCATGCTGTCACTG, SP6‐SCG10 anti‐sense primer: GGGATTTAGGTGACACTATAGAA‐AGCAGCTAGATTAGCCTCACGGT) were transcribed in the presence of UTPαS (1000–1500 Ci/mmol [35S] specific activity, Perkin Elmer). [35S]‐labeled probe was applied to the brain sections at a concentration of 2 × 104 cpm/mm2. Two adjacent sections per subject were studied at the level of the caudal hippocampal fold. Slides were hybridized at 55°C overnight and apposed to Imaging Plates (Fujifilm) along with [14C] standards (American Radiolabeled Chemicals). Films were developed with a FLA‐7000 phosphoimaging analyzer (Fujifilm). Images were analyzed using MultiGauge software (Fujifilm). Relative mRNA expression levels were measured along the CA1‐CA3 subregions of the hippocampus. Values from duplicate brain sections for each subject were averaged and expressed as dpm/mg of tissue by reference to co‐exposed standards. Background normalization was performed on each section, and set to subcortical white matter area lacking SCG10 mRNA expression.

Statistics

iTRAQ data were statistically evaluated by the Wilcoxon rank‐sum test after log transformation of unscaled input data exceeding a cut‐off of ± 2× geometric s.d. relative to vehicle‐treated controls. The fEPSP slope was expressed as a percentage of the average of 10‐min baseline preceding LTD induction. In both data from electrophysiology studies and the cohort of human fetal samples for mRNA analysis, the Shapiro–Wilk and Levene tests failed to demonstrate normal distribution and equality of variances, respectively. Thus, non‐parametric Mann–Whitney U‐test (independent samples) was chosen for statistical analysis. The mRNA expression levels (dpm/mg) from human fetal brains were normalized by natural log transformation, and plotted (ln(dpm/mg); Fig 4A1 and B). To analyze both SCG10 mRNA and protein expression in human tissues, general linear model analysis was used to control for the effects of other confounding factors alongside cannabis exposure. Variables were clustered as (i) fetal age, (ii) fetal body weight and foot length (“developmental measures”) and (iii) cannabis exposure. A univariate model was first performed with each cluster of variables and any variable that showed P <0.10 was included in the stepwise regression in addition to cannabis exposure (Hurd et al, 2005). Variables with P <0.05 were included in the final regression model as covariates of cannabis exposure. In all other experiments, group comparisons were performed by Student's t‐test unless otherwise stated. Results of acute pharmacological experiments on organotypic slices were analyzed using one‐way ANOVA followed by Tukey's post‐hoc comparisons. A P level of < 0.05 was considered statistically significant (SPSS 21.0). Data were expressed as means ± s.e.m.

Acknowledgements

This work was supported by the Scottish Universities Life Science Alliance (T.Har., A.A.), Vetenskapsrådet (T.Har.), Hjärnfonden (T.Har.), Novo Nordisk Foundation (T.Har.), the Wellcome Trust (equipment grants, C.H.B.), the Petrus and Augusta Hedlunds Stiftelse (T.Har.) and the National Institutes of Health (RO1‐DA023214, T.Har. & Y.L.H.; F31‐DA031559, C.V.M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health. We thank M. Watanabe (Hokkaido University) for antibodies, E.T. Coffey (University of Turku) for a vector containing point mutated EGFP‐SCG10S62D/S73D, M. Zilberter (Karolinska Institute) for his contribution to the initial phase of electrophysiology experiments, O.K. Penz (Karolinska Institutet) for technical assistance, I. Parisi and J. Mulder (Science for Life Laboratory) for assistance with automated slide‐scanning microscopy, and H.‐C. Lu (Baylor College), I. Galve‐Roperh (Complutense University), T. Hökfelt (Karolinska Institutet) and members of the Harkany laboratory for constructive discussions and feedback. Laser‐scanning microscopy was performed at the Click Imaging Core Facility of the Karolinska Institutet.

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